TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells-3

<p><b>Copyright information:</b></p><p>Taken from "TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells"</p><p>http://www.biomedcentral.com/1471-2202/8/49</p><p>BMC Neuroscience 2007;8():49-49.</p><p>Published online 4 Jul 2007</p><p>PMCID:PMC1931605.</p><p></p>depicted. () TRPM5-expressing cells are abundant in the cardiac region of the stomach. In gut epithelia, TRPM5-enriched cells are detected in the duodenal villi (), the ileum () and the colon (). Scale bars are 20 μm

B2R do not induce CCR2 despite NFAT activation.

<p>(A) BAM8-22- (2 µM) or BK-induced (1 µM) calcium signals were determined in single fura2-loaded F11-MRGPR-X1 cells by calcium imaging. Data of ∼300 cells were compiled and expressed as the mean ± S.E.M. RTQ-PCR experiments were performed with cDNAs derived from serum-starved F11-MRGPR-X1 cells stimulated or not with BK (1 µM) for 1 h in (B) or 6 h in (E). Relative BK-induced gene expression was normalized to β-actin, calculated using the ΔΔCp method, and expressed as the mean ± S.E.M. (C) Serum-starved F11-MRGPR-X1 cells were stimulated or not with BK (1 µM) for the indicated period of time and expression of EGR-1 was determined by western-blotting. Afterwards blots were stripped and re-probed with an antibody against ERK-2 (t-ERK-2). One representative blot is shown. Ligand-induced EGR-1 expression was quantified by densitometry and is given normalized to not stimulated cells as the mean ± S.E.M. (D) BK-induced (1 µM, 6 h) activation of the NFAT reporter is shown in F11-MRGPR-X1 cells. Data are expressed as the mean ± S.E.M. PD-184352 (10 µM, 30 min) was used to inhibit ERK-1/2 activity in (B) or CsA (1 µM, 30 min) to block calcineurin in (D and E). In (B–E) 4 independent experiments were conducted, respectively. In (F) CCR2 protein expression in F11-MRGPR-X1 cells was assessed by CCL2-promoted (100 ng/ml) inhibition of FSK-induced (5 µM) cAMP accumulation after pre-stimulation of the cells with BAM8-22 or BK (1 µM, 20 h). In (F, left panel) one representative experiment is shown and in (F, right panel) data from 5 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BK-stimulated inhibitor-treated and untreated cells or between BK- and BAM8-22-stimulated cells.</p

TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells-5

<p><b>Copyright information:</b></p><p>Taken from "TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells"</p><p>http://www.biomedcentral.com/1471-2202/8/49</p><p>BMC Neuroscience 2007;8():49-49.</p><p>Published online 4 Jul 2007</p><p>PMCID:PMC1931605.</p><p></p> lysates of mock- (line 1), TRPM5- (line 2) and YFP-TRPM5- (line 3) transfected HEK 293 cells. Positions of TRPM5- and YFP-TRPM5-specific bands are indicated by arrowheads. () Overlay of confocal and corresponding differential interference contrast (DIC) images of immunofluorescence staining of HEK 293 cells transiently expressing wild-type TRPM5. () Immunohistochemistry of taste buds using a TRPM5-specific antibody at lower (C) and higher magnifications (D, E). Notably, TRPM5 is predominantly localized on the basolateral surface of cells (indicated by the arrow in (D, E)). () Lack of TRPM5 immunoreactivity in taste receptors after preincubation of the TRPM5-specific antibody with the immunization peptide. () Fluorescent labeling of taste buds with the TRPM5-specific antibody (E) and the UEA lectin (H) and overlay of both with corresponding DIC image (I). Note that only a subset of cells in the taste bud is enriched in TRPM5. In the TRPM5-positive cells, TRPM5 is mainly localized on the basolateral cell surface and absent in microvilli (as indicated by an arrow (G, H, I)). Scale bars are 20 μm each

TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells-1

<p><b>Copyright information:</b></p><p>Taken from "TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells"</p><p>http://www.biomedcentral.com/1471-2202/8/49</p><p>BMC Neuroscience 2007;8():49-49.</p><p>Published online 4 Jul 2007</p><p>PMCID:PMC1931605.</p><p></p> () Immunolocalization of TRPM5 in solitary cells of the main olfactory epithelium is shown at lower (indicated by the arrows in (A, B)) and higher magnifications (C, D). () Immunohistochemistry of TRPM5 in the VNO. (E, F) Localization of TRPM5 on the apical surface of sensory epithelia (indicated by the arrow). (G, H) Detection of TRPM5-enriched solitary cells in the non-sensory part of the VNO. Scale bars are 20 μm

TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells-4

<p><b>Copyright information:</b></p><p>Taken from "TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells"</p><p>http://www.biomedcentral.com/1471-2202/8/49</p><p>BMC Neuroscience 2007;8():49-49.</p><p>Published online 4 Jul 2007</p><p>PMCID:PMC1931605.</p><p></p> corresponding DIC images (right panels) are shown. The TRPM5-enriched cells in gastric glands of mouse stomach (A, B) and in mouse a duodenal villus (C, D) reveal a long basolateral processes (indicated by the arrows). Within duodenal epithelia (C, D), the TRPM5-immunoreactive cell contains a protrusion extending beyond the epithelial luminal border (indicated by an arrowhead). () Co-localization of TRPM5 with CK18 in intestinal glands of the rat duodenum. Confocal images of TRPM5 and CK18 specific signals (E, F) and their overlay with corresponding DIC images (G) are depicted. Scale bars are 10 μm

MRGPR-X1 induce EGR-1 via ERK-1/2 and CCR2 via NFAT in primary DRG neurons.

<p>BAM8-22-induced (2 µM) calcium signals in rat DRG neurons co-expressing MRGPR-X1 and aequorin or solely aequorin are presented in (A). BAM8-22-induced (2 µM, 8 h) activation of the NFAT (B) or TCF/SRF (C) reporter is shown in rat DRG neurons transiently co-expressing MRGPR-X1. RTQ-PCR experiments were performed with cDNAs derived from serum-starved MRGPR-X1 expressing rat DRG neurons stimulated or not with BAM8-22 (2 µM) for 6 h (D) or 40 min (E). CsA (1 µM, 30 min) was used to block calcineurin in (B and D) or PD-184352 (10 µM, 30 min) to inhibit ERK-1/2 activity in (C and E). Relative BAM8-22-induced gene expression was normalized to β-actin and calculated using the ΔΔCp method. Data from 4 independent experiments were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells-0

<p><b>Copyright information:</b></p><p>Taken from "TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells"</p><p>http://www.biomedcentral.com/1471-2202/8/49</p><p>BMC Neuroscience 2007;8():49-49.</p><p>Published online 4 Jul 2007</p><p>PMCID:PMC1931605.</p><p></p> lysates of mock- (line 1), TRPM5- (line 2) and YFP-TRPM5- (line 3) transfected HEK 293 cells. Positions of TRPM5- and YFP-TRPM5-specific bands are indicated by arrowheads. () Overlay of confocal and corresponding differential interference contrast (DIC) images of immunofluorescence staining of HEK 293 cells transiently expressing wild-type TRPM5. () Immunohistochemistry of taste buds using a TRPM5-specific antibody at lower (C) and higher magnifications (D, E). Notably, TRPM5 is predominantly localized on the basolateral surface of cells (indicated by the arrow in (D, E)). () Lack of TRPM5 immunoreactivity in taste receptors after preincubation of the TRPM5-specific antibody with the immunization peptide. () Fluorescent labeling of taste buds with the TRPM5-specific antibody (E) and the UEA lectin (H) and overlay of both with corresponding DIC image (I). Note that only a subset of cells in the taste bud is enriched in TRPM5. In the TRPM5-positive cells, TRPM5 is mainly localized on the basolateral cell surface and absent in microvilli (as indicated by an arrow (G, H, I)). Scale bars are 20 μm each

MRGPR-X1 induce CCR2 via NFAT in F11-MRGPR-X1 cells.

<p>RTQ-PCR experiments were performed with cDNAs derived from serum-starved F11-MRGPR-X1 cells stimulated or not with BAM8-22 (1 µM) for 6 h (A-C) using specific primers for 5 distinct genes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058756#pone-0058756-t001" target="_blank">table 1</a>. The name of the analyzed gene is listed beneath the corresponding bar. Genes with a p value <0.05 are show in black bars. In (B and C) cells were treated or not with CsA (1 µM, 30 min). Relative BAM8-22-induced gene expression was normalized to β-actin and calculated using the ΔΔCp method. Data from 4 independent experiments were compiled and expressed as the mean ± S.E.M. BAM8-22-induced (1 µM, 20 h) CCR2 protein expression in F11-MRGPR-X1 cells was assessed by flow cytometry (D) or by CCL2-promoted (100 ng/ml) inhibition of FSK-induced (5 µM) cAMP accumulation (E). In (D) one representative experiment is shown. Accumulation of the data from 5 independent experiments revealed an increase in the number of CCR2 positive cells by 10.8±1.4% after BAM8-22 stimulation. In (E, left panel) one representative experiment is shown and in (E, right panel) data from 5 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

MRGPR-X1-induced signaling in DRG neurons and CTMCs.

<p>A cartoon illustrating the signaling circuit by which MRGPR-X1 affect chemokine signaling in DRG neurons and connective tissue mast cells is given.</p

MRGPR-X1-induced CCL2 release in LAD-2 mast cells.

<p>(A) MRGPR-X1 (40 cycles) or β-actin (30 cycles) mRNA expression was determined in LAD-2 cells by RT-PCR. As a negative control, RT-PCR was conducted without addition of cDNA (H₂O). (B) Calcium signals in fura2-loaded LAD-2 cells are shown after injection of BAM8-22 (5 µM) or ionomycin (5 µM) or HBS as positive or negative control, respectively. (C) CCL2 release in LAD-2 cells after stimulation with BAM8-22 (5 µM, 18 h) was determined by ELISA. Data from 4 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells.</p